Crb1 is found to be normally expressed in many tissues, including lymph node, cortex, cerebellum, olfactory bulb and the retina, and also during development. Crb1 is thought to be involved in maintaining the integrity of the external limiting membrane and mutations in Crb1 result in genetic retinal disorders such as retinitis pigmentosa and Leber congenital amaurosis in humans, den Hollander et al. 2001, Am J Hum Genet 69:198-203.
The retinal degeneration 8 (rd8) mutation of the Crb1 gene (Crb1rd8; Crb1<rd8>) is a frequently found in inbred mice, being present in ˜20-25% of background. This mutation is a loss of a single nucleotide in exon 9 leading to a frame shift and early termination of protein translation that truncates the transmembrane and cytoplasmic domain of the Crb1 protein. The frame shift introduces a new stop codon (TGA) 144 bp downstream from the rd8 nucleotide deletion, Mehalo et al. 2003, Hum Mol Genet, 12(17):2179-89. Mutations in Crb1 in mice lead to visual abnormalities (Bulgakova & Knust 2009, 122, 2587-2596). The Crb1rd8 mutation results in irregular retinal lesions in the inferior nasal quadrant of the fundus and the Crb1 complex is also implicated in the regulation of renal epithelia polarity, Pieczynski & Margolis, 2011 (Am. J. Physiol. Renal Physiol, 300(3): F589-F601), and tumorigenesis, Laprise, 2011 (J Biomed Biotechnol, 2011; 2011:868217. doi: 10.1155/2011/868217).
One of the inbred mouse strains that carry the Crb1rd8 mutation is the mouse strain C57BL/6N, lacking a C nucleotide in the Crb1 gene. This has implications for all ocular vision research models on C57BL/6N background. With the C57BL/6N embryonic stem (ES) cell line as a major workhorse for genetic engineering to create new mouse models, the mutation in Crb1 impacts the downstream phenotypic analysis. The issue of the rd8 mutation of the Crb1 gene and impact on the phenotypic analysis has been recently discussed in Mattapallil et al. 2012, Invest Ophthalmol Vis Sci 53:2921-7; Luhmann et al. 2012, PLoS One 7:e35551.
Outcrossing of this mutation by congenic backcross approaches would be cumbersome, time-consuming and expensive, and as with all backcrossing it would never be guaranteed that such approach would be successful, as backcrossing is limited by recombination, and may never be complete, as it has been reported that certain DNA stretches persist.
Methods and compositions are provided by the present invention for correcting a mutation in the Crb1 gene of mice characterized by non-functional Crb1 protein. The present invention provides genetically modified animals and cells including edited chromosomal sequences encoding a corrected Crb1rd8 mutation. In particular aspects of the present invention, the genetically modified animals or cells including edited chromosomal sequences encoding a corrected Crb1rd8 mutation are generated using a Transcription Activator-Like Effector Nuclease (TALEN).